We have identified a novel physiological anti-inflammatory pathway under the control of the transcription factor Erg; this pathway inhibits NF-κB-dependent transcription and TNF-α-induced inflammation in vivo. These results suggest a novel approach to anti-inflammatory therapies.
Regions of the arterial tree exposed to laminar flow, which exerts high shear stress, are protected from inflammation, endothelial cell (EC) death and atherosclerosis. TNFalpha activates NF-kappaB transcription factors, which potentially exert dual functions by inducing both proinflammatory and cytoprotective transcripts. We assessed whether laminar shear stress protects EC by modulating NF-kappaB function. Human umbilical vein EC (HUVEC) were cultured under shear stress (12 dynes/cm2 for 16 h) using a parallel-plate flow chamber or were maintained in static conditions. Comparative real-time PCR revealed that preshearing significantly alters transcriptional responses to TNFalpha by enhancing the expression of cytoprotective molecules (Bcl-2, MnSOD, GADD45beta, A1) and suppressing proinflammatory transcripts (E-selectin, VCAM-1, IL-8). We demonstrated using assays of nuclear localization, NF-kappaB subunit phosphorylation, DNA-binding, and transcriptional activity that NF-kappaB is activated by TNFalpha in presheared HUVEC. Furthermore, a specific inhibitor revealed that NF-kappaB is essential for the induction of cytoprotective transcripts in presheared EC. Finally, we observed that NF-kappaB can be activated in vascular endothelium exposed to laminar shear stress in NF-kappaB-luciferase reporter mice, thus validating our cell culture experiments. We conclude that shear stress primes EC for enhanced NF-kappaB-dependent cytoprotective responsiveness while attenuating proinflammatory activation. Thus modulation of NF-kappaB function may underlie the atheroprotective effects of laminar shear stress.
Background Although monocyte-derived tissue factor (TF) plays critical roles in atherothrombosis, little is known about its posttranscriptional regulation. Tristetraprolin (TTP) binds the 3′UTR of target mRNAs and promotes their degradation, with its function being negatively regulated by p38 MAPK. Whether TTP posttranscriptionally regulates TF is unknown. Methods We used human monocytes and bone marrow derived macrophages from TTP+/+ and TTP-/-mice. Procoagulant activity was determined using a clot turbimetric assay. mRNA decay was determined following transcriptional arrest using actinomycin D. TTP knockdown was achieved using siRNA transfection. RNA and protein interaction was determined using ribonucleoprotein (RNP) immunoprecipitation and RNA biotin pulldown assays. Results p38 inhibition with SB203580 and SB202190 (1µMM) reduced procoagulant activity, TF mRNA and protein expression in human macrophages (p<0.05). p38 inhibition reduced TF mRNA stability in both human and murine macrophages (p<0.05). TTP knockdown increased TF expression in human macrophages (p<0.05). Both TF mRNA and protein expression were significantly increased in TTP-/-versus TTP-/-macrophages (p<0.05). Moreover TF mRNA decay was reduced in TTP-/-macrophages and p38 inhibition had no effect on this (p<0.01). RNP immunoprecipitation demonstrated TTP and TF mRNA interaction. Furthermore, a more specific interaction with TTP and TF 3′UTR was confirmed using RNA biotin pulldown techniques. Conclusions These data provide evidence, for the first time, that p38 and TTP post-transcriptionally regulate TF expression in macrophages. A better understanding of the post-transcriptional regulation of TF expression will provide novel insights into the interface between inflammation and thrombosis in vascular biology, and holds therapeutic potential.
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